Peripheral blood monocytes are derived from bone marrow hematopoietic stem cells and are known to differentiate into several phagocytes, including macrophages, dendritic cells, osteoclasts, microglial cells of central nervous system, and liver Kupffer cells (Bioessays, 17, 977-986, 1995; Blood, 98, 2544-5254, 2001; BMC Immunol., 3, 15, 2002; Microsc Res Tech., 39, 350-364, 1997). The differentiation of the monocytes into various phagocytes is controlled by signaling of various growth factors. In other words, differentiation into macrophages, dendritic cells, osteoclasts are controlled by signaling of M-CSF, GM-CSF and IL-4, and receptor activating factors of NF ligand or M-CSF, respectively (see for example, Blood, 98, 2544-5254, 2001; BMC Immunol., 3, 15, 2002; J Exp Med., 190, 1741-1754, 1999). Until recently, it was believed that the differentiation potential of monocytes was restricted to phagocytes. However, recent studies have shown that human monocytes can differentiate into endothelial-like cells by culturing in vitro with a combination of angiogenic factors (see for example, Differentiation, 65, 287-300, 2000; Cardiovascular Res., 49, 671-680, 2001). In addition, the expression of bone-specific alkaline phosphatase was reported during the monocyte differentiation process in the in vitro granuloma model (see for example, Immunobiology, 202, 68-81, 2000). However, the differentiation potential is not completely clarified and it was not known whether monocytes had differentiation potential into cell types other than phagocytes.
On the other hand, it was revealed that many adult tissues contain populations of stem cells that can self-replicate and give rise to daughter cells that undergo an irreversible terminal differentiation (see for example, Science, 287, 1442-1446, 2000). The best-characterized are hematopoietic stem cells and their progeny, but stem cells are identified in most of the tissues, including mysenchymal, neuron, and hemotopoietic cells (see for example, Science, 284, 143-147, 1999; Science, 287, 1433-1438, 2000; J. Hepatol., 29, 676-682, 1998). Mesenchymal stem cells (MSCs) are identified as adherent fibroblast-like cells in the bone marrow with differentiation potential into mesenchymal tissues, including bone, cartilage, fat, muscle, and bone marrow stroma (see for example, Science, 284, 143-147, 1999). Recently, mesenchymal progenitors having morphologic and phenotypic features and differentiation potentials similar to MSCs have been reported at extremely low frequencies in umbilical cord blood (see for example, Br. J. Haematol., 109, 235-242, 2000), as well as in fetal (see for example, Blood, 98, 2396-2402, 2001) and adult peripheral blood (see for example, Arthritis Res., 2, 477-488, 2000). However, MSCs and circulating MSC-like cells do not express various hematopoietic markers or the stem cell/endothelial marker CD34 (see for example, Science, 284, 143-147, 1999; Br. J. Haematol., 109, 235-242, 2000; Blood, 98, 2396-2402, 2001).
As described above, various postnatal tissue-specific stem cells and embryonic stem (ES) cells are currently being analyzed as candidate sources for future therapeutic intervention for tissue regeneration (see for example, Science, 287, 1442-1446, 2000). It has been reported that bone marrow-derived MSCs engraft in many organs and differentiate along tissue-specific lineages upon its transplantation in animal models (see for example, Nat. Med., 6, 1282-1286, 2000; Science, 279, 1528-1530, 1998), as well as in human infant suffering osteogenesis imperfecta (see for example, Nat. Med., 5, 309-313, 1999). However, MSCs are rare in adult human bone marrow (0.01% to 0.001%), and expansion of MSCs to the number of cells required for regeneration therapy is technically difficult, expensive, and time-consuming (see for example, Stem Cells, 19, 180-192, 2001). ES cells are multipotent cells derived from germinal cells that can be propagated indefinitely in vitro being still undifferentiated and induced to differentiate to most cell types in vivo (see for example, Trends Biotechnol., 18, 53-57, 2000). Although ES cells have been isolated from human, their use in research as well as therapeutic is cumbered by ethical considerations (see for example, Science, 287, 1397, 2000).
Several different precursors that can differentiate into endothelial or mesenchymal cell types have been reported in human postnatal peripheral blood, including endothelial cells (see for example, Science, 275, 964-967, 1997), smooth muscle cells (see for example, Circulation, 106, 1199-1204, 2002), and mesenchymal cells (see for example, Arthritis Res., 2, 477-488, 2000). In vitro expansion of endothelial and smooth muscle progenitors requires a combination of several growth factors (see for example, Science, 275, 964-967, 1997; Circulation, 106, 1199-1204, 2002). Mesenchymal progenitors can be expanded in a medium supplemented with 20% fetal bovine serum (FBS) without any additional growth factors, but their development in PBMC cultures was reported to be unaffected by eliminating CD14+ cells (see for example, Arthritis Res., 2, 477-488, 2000). However, these endothelial or mesenchymal cells do not have the phenotypic characteristics to be positive to CD 14, CD45, CD34 and type I collagen.
The big object remaining in modern medicine is said to overcome deficiency of organs due to disease or external injuries or functional impairment. The only method that can be practiced today for treating such condition is organ transplantation. However, there are still many difficulties for spreading as an actual treating method, due to problems such as brain-death diagnosis or supply from donors. On the other hand, regenerative medicine intending regeneration of organs draws attention with the recent development of stem cells and developmental biology, and is expected as the direction of the medicine to advance in the 21st century. In animal models, functional recoveries of organs by transplantation of ES cells have been reported, while the application in human is stuffed due to rejection or ethical problems of the use of ES cells. Further, as various adult tissues stem cells (mesenchymal, blood vessels, liver etc.) are extremely few in vivo, the isolation thereof is technically difficult, and it is hard at the present time to obtain sufficient amount of cells for transplantation. Therefore, there are many problems to be solved before the regenerative medicine using ES cells or tissue stem cells can be applied to the actual medicine. Particularly, it is essential to supply cells having differentiation potential in a stable manner so that regenerative medicine becomes a reality.
The object of the present invention is to provide a multipotent cell that can differentiate into various cells such as mesenchymal cells including bone, cartilage, skeletal muscle and fat, endothelial cells, myocardial cells and neurons wherein a sufficient amount can be supplied stably with minimum invasion, without problems such as securing donors and rejection in cell transplant, and with less ethical considerations; a mesodermal progenitor/mesodermal cell/mesodermal tissue and a neuron/nerve tissue, such as mesenchymal cells, myocardiac cells, endothelial cells, being induced to differentiate from the multipotential cell; a therapeutic agent comprising these as active ingredient; and a treating method administering the same.
The present inventors confirmed the expression of fibroblast-like cells when peripheral blood mononuclear cells (PBMCs) are cultured on fibronectin-coated plastic plates for 7 to 10 days. Being interested by the origin and physiological function of this human cell population exhibiting a fibroblast-like morphology, the present inventors found that these cells are derived from circulating CD14+ monocytes, with a unique phenotype of CD14+CD45+CD34+ type I collagen+, and having a potential to differentiate into mesenchymal cells including bone, cartilage, smooth muscle and fat, endothelial cells, myocardial cells, neurons, under a particular culture condition. They named this cell the monocyte-derived multipotent cell (MOMC). With the knowledge having revealed for the first time that circulating monocytes are multipotent progenitors having a differentiation potential not only into phagocytes but also into various mesenchymal cells, the present inventors have thus completed the invention.